Development of envelope protein immunogens capable of eliciting neutralizing antibodies against primary HIV-1 isolates from different genetic subtypes remains the most difficult challenge in the field of HIV-1 vaccinology. The conserved membrane-proximal external region of the gp41 ectodomain bears the epitopes of two broadly neutralizing human monoclonal antibodies, 2F5 and 4E10, and is therefore an important target of HIV-1 vaccine design. However, all attempts to elicit anti-gp41 antibodies comparable to these produced in infected humans by using unstructured peptide immunogens have thus far failed. Success will likely require a deeper understanding of the structural motifs of the gp120/gp41 complex that stabilize the mature envelope trimer on the virion surface. Current thinking postulates that the receptor-mediated activation of gp41 involves a regulated sequence of structural transitions with one or more on-pathway intermediate(s) that may reveal cryptic neutralization epitopes. Our recent identification of a four-stranded coiled-coil structure (C43) encompassing the 2F5 epitope suggests that structural aspects of the 2F5 and 4E10 epitopes may be transiently exposed and required for stimulating neutralizing antibody responses. The broad, long-term objective of this research plan is to use protein chemistry and structural biology approaches to engineer and produce stable forms of the C43 coiled-coil motif for the induction of broadly reactive neutralizing antibodies. The premise of the proposed research is that definition of the structural requirements for optimal presentation of the 2F5 neutralization epitope can lead to the identification of stable immunogen products to generate effective anti-HIV-1 immunity. The Specific Aims of the proposed research are: (1) To design and develop stabilized versions of the C43 coiled-coil domain bearing the 2F5 epitope for immunogenicity studies. We will generate a single-chain analog of the tetraplex coiled coil in which the four mutagenesis to identify specific residue substitutions that favorably influence inter-helical packing interactions in the tetramer in order to stabilize an ordered helical peptide structure of the 2F5 epitope that preserves surface-exposed side chains. (2) To evaluate the immunological responses elicited by stabilized C43 coiled-coil variants in small animals. We will conduct immunogenicity studies in rabbits and guinea-pigs to determine whether the stabilized involve immunization of the animals using the stabilized C43 peptide proteins. We will also evaluate the immunogenicity of the stable coiled-coil molecules captured onto nanometer-sized beads as particulate immunogens. The research and public health communities concur that a preventive vaccine is the obvious long-term solution to bring the global HIV-1 epidemic under control (1-8). Unfortunately, this goal has proven elusive and no such vaccine is available. Overcoming this important biomedical problem will require new and imaginative design strategies to bring us closer to the goal of a successful AIDS vaccine.